Utilizing the genetically tractable model system of the zebrafish, Danio rerio, we will investigate the molecular mechanisms that control craniofacial cartilage morphogenesis. This proposal is based on the hypothesis that RhoA and its targets are the effectors of the noncanonical Wnt/PCP pathway controlling chondrocyte stacking in zebrafish. To investigate this hypothesis we will determine 1) the localization of RhoA activity within the cells of the developing cartilage, 2) if Rho kinase (Rok2), a target of RhoA, plays a role in the control of chondrocyte stacking downstream of Knypek and WntSb and 3) if Rok2 inhibits Ppp1r12a as part of the noncononical Wnt/PCP pathway that effects chondrocyte stacking. We predict that RhoA will act downstream of the Glypican4/Knypek and Wnt5b/Pipe tail controlled Wnt/PCP pathway in a polarized, asymmetric fashion. While RhoA is a well-established member of the Wnt/PCP pathway in other cellular contexts, it has not been identified to take part in the control of chondrocyte morphogenesis. By utilizing the optically clear embryos of zebrafish, and novel imaging techniques we will determine the subcellular localization of RhoA activity within the developing chondrocytes of wild type and well-characterized mutant zebrafish embryos. To investigate the potential role of Rho kinase2 within the process of cartilage morphogenesis, we will attempt to suppress the glypican4/knypek or wnt5b/pipe tail mutant phenotypes through the overexpression of an activated form of Rok2. Finally, we predict that the actin cytoskeleton plays a central role in regulating the shape and behavior of chondrocytes and does so through Ppp1 r12a, an inhibitor of the myosin light chains. We will establish if Ppp1 r12a helps control chondrocyte behavior by determining if a genetic interaction exists between ppp1r12a and glypican4/knypek or wnt5b/pipe tail through the creation of double homozygous mutants. Craniofacial abnormalities pose an enormous burden to the individual, their families and the health care system as a whole. This proposal will add to our understanding of the mechanism used to properly develop craniofacial cartilage. By utilizing zebrafish as a model to determine how normal cartilage forms we can gain insight into the potential cause of many craniofacial defects which in turn may lead to new diagnostic and therapeutic techniques to treat these abnormalities.